A METHOD FOR THE BIOLOGICAL CONTROL OF POLLEN IN PLANTS

Abstract: A method for the biological control of pollen in plants, including the thinning of fruits, reducing seeds in citrus plants and reducing the incidence of shotberries in grapes, by applying an effective amount of activated or inactivated RNase or mixtures of these to the flowers of the plants.

Description: A METHOD FOR THE BIOLOGICAL CONTROL OF POLLEN IN PLANTS BACKGROUND OF THE INVENTION The present i nven t i on re l a t es t o a met hod of the biological control of pollen in plants using RNase. The present invention particularly relates to the thinning of a large variety of fruit plants, the reduction of seeds in ci trus plants and the reduct ion of the incidence of shotberrie in grapes using activated RNase, inactivated RNase or mixtures of these. The purpos o f thi nning f rui t , whether i t i s done manually, chemically or mechanically, i s to reduce crop irly in the frui t development period to maximize from clone to clone, and climatic diminish the efficiency of the chi nectarines hand thinning i s the only pract ical method that is current ly employed, simply because none of the Xiloyannis, J . Am. Soc . Hor t . Sci . , 40. ( 1976 ) , pp 237-247, and G. E. Stembridge, C. G. Gambrel l , Ibid 96 , (1971) pp. 7-9), urea, s well as cold water. T o major problems arc associated with these methods: 1. Thinning intensi y was inconsistent and difficult to con t τo 1. 2. The broad range of ef fect makes i t impossible to determine a preci se development al s t age (of the flower or the fruitlet) for optimal treatment In grapes , g i bberel l ic acid i s rout inely used or thinning To pson cultivar. The results, however, require always hand thinning for adjustment. The same treatment in Perlette is not practiced for the last 15 years, since it was found that in this variety, the gibberellic acid causes irregular berry size (shortberries ) , therefore hand thinning is practiced in Pt'rlet le. The series of events that occur between pollination and fertilization include processes of specific recognition of pollen grains and pollen tubes by the pistil tissues. The control of t h pi t i l on pol len ger inat ion and po l l en t ube g r ow t h ha s been ma i n l y s t ud i ed i n se 1 f - i ncompa t i bl c sys t ems , in whi ch arres t of pol len tube is the consequence of a contact bet een ollen and istil of the me genotype. In self-compatible matings the e is some evidence t at the pollen tube is directed toward the ovule by trophic and chemotropic ef fect in t he s tyle . H ever , there i s s t i l l a auci ty of inf orma t i on abou t the f e a l e con t rol of th ma le g aine t ophy t e . Most economically important apple ( ( alus domes t ica) cul tivar.. require cr o s s -pol 1 i na t i on for fruit set. Peach (Prunus persica) is known as self- compatible. In these species the growth of pollen tubes in the s ty le ha een t o ou hly inves t i at ed by physiologic l and hi stochemical studies . I t has been established that the kinetics of pollen tube growth is controlled by each part of the gynoecium, starting with the st igma and en ing i l l, the ovule. However , the nature of this control , or any stylar product in which it i accomplished by, have not been characterized in these wor s . Recently a major stylar protein (S-protein) was isolated from the self-incompatible species Nicot i ana alata. The gene tha t encodes f or t he protein was c loned and sequenced and wa found to sha homology wi th fungal RNase. It was established that this protein i s indeed RNase, and was found to arrest pollen tube growth (B. A. McClure et al. , Nature, 1989, pp. 955-957). However, the RNase was al so found in s t yl ar di f fusates of sel f- compatible species such as Ni co t i na tabacum (McClure et al. , 1989) and more recently in peach (Roiz and Shoseyov, 1993, unpublished data) and citrus (Roiz, Shoseyov and Goren, 1993, unpublished data). I t i s speculated that the RNase penetrate the pollen tube, degrade riboso al and RNΛs and thereby cause its abortion. Closely related to the thinning of the frui t s is the reduct ion of seeds in ci t rus lant s , hic i s very desirable from a practical as well as commercial point of view. Unti l no there i s no known eliable method for reducing the seeds in citrus plants. Similarly, there is no known reliable method for reducing the incidence of shotberry in grapes. RNase is a known enzyme obtainable from a large variety of sources. One ain source i s the fermentat ion of various fungi. SUMMARY OF THE INVENTION In accordance i th the present invent i on there i s provided a method for the biological control of pollen in plant , compris ing applying an ef fec t ive amount of activated or inactivated RNase or a mixture thereof. The resent invent ion resul t s in the thinning of a large variety of fruit plants, the reduction of seeds in citrus plants and, in the case of grapes, the reduction of the incidence of shotberries. DET ILED DESCRI P ION OF THE INVENTION RNase m y be obtained from sources s ch s fungi , bacteria, plants, animal and viral . When obtained from fungal sources, the RNase may easi ly be obtained by the fermentation of various fungal sources. Examples are Λ per i l lus niger , Λspergi l lu clari tus, Aspergi l lus oryzae and Rhisopus iveus, preferably Aspergillus niger and Aspergil lus oryzae. A mos t preferred fungus i s Aspergillus niger BKCMICC Number 324,626). This strain wa s depo si ted w i t h t he Commonwea l t h Mvco l og i c a l Institute, Ferry Lane, Kew, Ri chmond-upon-Thame s , Surrey TW9 3Λ5, Uni ted Kingdom, on 20 May, 1988. The plants where the method of the biological control of pollen is chosen from the grou cons i s t in of frui t trees, orna ent al t e , vege t ables , f i eld crops , plantations, ornamentals and the like. The trees where the m thod of thinning was found to be applicable are chosen from the group consi s t ing of deciduous plants, tropical plants, sub-tropical plants, citrus plants, nut tree and grape vines. Examples of deciduous plants are stone fruit , pears , apples , quince, peaches , plums , nectarines, cherries and apricots. Examples of ornamental trees are olive and Fica trees. Exam les of field cr are cereals, especially hybrids. Examples of ci trus plant where the RN s in use in t e thinning and the reduction of seeds are orange, lemon, grapefruit, Clementina , and rin, ci tron, pomela nd their hybrids. The method of the prevention of frui t set of field crops of the resent invent ion was found to be especial ly useful in preventing fruit set of ornaments whose fruits and flowers cause environmental problems. The RNase is added to the pollen in the form of crude or purified e t c t of 11.e RNase in the or of spr y, powder and the like. A really novel way to apply he RNase is to powder bees and let them distribute the RNase powder during their feeding on the pollen in the flowers. ■The RNase is applied at a rate of 1 to 10,000 uni t s per hec tare. Whi le the invention wi ll now be described in connection ith certain preferred embodiment s in the fol lo ing examples, i t wi ll be understood that i t i s not intended to limi t the invent ion to these part icular embodiment s. On the contrary, i t i intended to cover al l a l terna¬ tives, modi ficat ions and equivalent s, as may be included wi thin the scope of the invent i on as defined by the appended claims. Thus , the fol lowing examples , which include referred embodiments, i l l serve to i llustrate the practice of thi s invention, i t being understood that the particul ar s shown are by way of exampl e and for purposes of i llustrative discussion of preferred embodi¬ ments of the present invention only and are presented in the cause of r viding hat is bel ieved to be the most useful and readily understood description of procedures, as well as of the principles and conceptual aspect s of the invention. EXAMPLE 1 Growth Condi t ions Of Aspergi l lus ni ger Aspergillus niger was grown in medium that contained 1% wheat flour and 0.059. (NIU)?.S0 4 in disti l led water. The solution was brought to pll 3.5 wi th HC1 and then auto- claved. An inoculum of 10 6 spores of A. niger were sus¬ pended into 100 ml of t lie growth medium and the suspen¬ sion was incubated at 30°C and shaken at 150-200 rpm. RNase act ivi ty i n t lie rowth medium reached i t s peak after 5-7 days o incubation. The medium was then passed through a illipore fi l ter of 0.2 μm to remove any traces of hypha and spores . The medium was u 1 t r a f i 1 t e r ed to concentrate the enzyme. EXAMPLE RNa s e Ass v s RNase act ivi t y in t he rowth medium of A. ni ger was deter ined by di ffu ioti plate assay, using 0.1% torula yea t RNA (Sigma) in 1% agarose as a substrate. A drop of 10 μl was appl ied on the substrate in a 15cm petri dish and t hen w s i ncuba t ed a t 37 ° C for one hour . The substrate was then stained i th 0.02. toluidine blue in water. RNase activi ty was indicated as a whi te halo on a blue background. ■ A quant i tat ive spec t ropho t ome t r i c assay of RNase was conducted as a modification of the procedure described by Brown & Ho (1986) . Each sample contained lOOμl of the examined enzyme mixed with and 4 μg torula yeast RNA and 1 ml of 20 M buffer of choice. The sample was incubated in 37° for 30 minutes. The reaction was s topped by putting the tube into ice and adding 200 μl "stop reagent" (0.7.. < C uranyl sulphate in 2 1 IIClC ). The solution was centrifuged in 12,000 rpm for 5 minutes. The supern tant was di luted to 1/20 in disti lled water and OD in 260 nm was examined. The blank solut ion cont ained th sa e component and incubated by the sa e procedure, o ly the enzyme was added after the reaction was stopped. The resul ts were given in uni t s/ ml , e uivalent to standards of bovine pancreatic RNase. EXAMPLE 3 OpI imizat ion of R a e A tivi ty The opt imal pll for RNase act ivi ty in the growth medium was examined u ing 20 mM citrate buffer at the pH value of: 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0. The above spectrophoto etric procedure showed that the optimal pH for the fungal RNase act ivi ty was 3.5 (Fig. 1) . The effect of presence or lack of metal ions on RNase activity was also examined. Each of the following salts: MgCl 2 , FeCl 3 or CaCl2 was supplemented into the reaction mixture to a final concentration of 10 m . Addit ional samples cont ained t e ame concentrat ion of DTT as a reductant and EDTΛ that has trong affini ty to metal ions. RNase act ivi ty was examined by the above diffusion plate assay. No difference in the diameter of the white halo (ICDI) on the blue background was observed between the different treatments and the control. EXAMPLE A The Effect of RNase on Pollen Germination an Pollen Tu e Growt h in Vitro Pollen grains of peach cv. Texas were germinated in vitro in growth medium containing 1 % (w/v) sucrose, lOOμg/ml II3BO1, 200μg/ml MgSO-ι .7H 0 and 200μg/ml Ca ( NO 3 ) 2.4 II 2 0 (Galletta, 1983). In each 1.5 ml Eppendorf approximately 1000 pollen grains were suspended by the edge of a needle into 100 microl i ter s of growth medium. RNase was suspended in t he pol len gro th mediu to a f inal concentration of 0.05, 0.5, 5, 50 or 500 units/ml. The percentage of ger ination was examined after two hour of incubation at 25°C in the k and the length of the pollen-tubes was measured after incubat ion overnight at t he s a e condi t i ons . Table 1 s o s tha t the RNase produced by A. niger lias a clear inhibi tory effect on both pollen germination (P<0.02) and pollen tube growth (P<0.01). For a comparison, the results obtained from the same conditions with 5 units/ml bovine pancreatic RNase are also demonstrated. EXAMPLE 5 Fi l E pe im nt The RNase produced by A. niger was examined in t e field as a thi ning agent of deciduous fruit trees. In peach (Prunus persica) and plum (Prunus salicina) the fungal RNase was applied exogenously directly on the stigmas, by spraying the flowers during anthesis. The flowers were exposed to open pollination and the fruit-set was e mined our wee after the blooming period. In trees of peach cv. Texas grown in an orchard in Kefar Varbur d i fferent ranches were treated as foilow : - The flowers were left untreated (control) - The flowers were sprayed by 10 mM citrate buffer pll 3.5 just once, h n the percentage of open flowers was estimated as at l ast 50% (B-I) The same buffer was used, only spraying procedure was repeated every 2-3dayε, starting from 10% open flowers till the branch ceased blooming (B-M). The flowers were sprayed by RNase t at was diluted by t he above buf fer to 1 100 uni t s / l . RN a s e as ap lie onc (Rl -I and R1 0- I , respec t i ve l y ) or repeatedly (Rl-M and R100-M respec t ively) uring an t hes i s . Table 2 shows that RNase caused a significant reduction of about 40% ( I* < 0.01 ) of frui t-set . The repeated treatments showed better results than one treatment but interestin ly, the low concentration was most effective in t is case. TABLE 1 POLLEN GERMINATION AND POLLEN TUBE LENGTH IN DIFFERENT CONCENTRATION OF RNASE RNase Cone. Number of Pollen Pollen tube (uni t /mi 1 i 1 i ter Repetitions Germination length (mm) % 0 (contro1 ) 77.22±2.9i 0.57+0.10 0.5 81.05+8.95 10 0.56±0.14 50.63±0.62 17 0.33+0. 8 5" 42.89±2.97 55 0.31+0.08 a = pancreatic RNase. TABLE 2 FRUTT SET OV PEACH CV TFXAS IN KEFAR VARBURG Treatment Number of Total Percentage P=0.05 branches number of of fruits f 1 ower s er branch Con t ro 1 166 B-I 188 ab B-M 186 Rl-I '.] 4 be Rl-M R100-I 7 160 cd R100-M 7 217 bed In nectarine cv. Fantasia in Rosh-Zurim the effect of the pH of the solvent and the ef fect of Tri ton x-100 as a surfactant were examined. All the branches were sprayed repeatedly, starting from 10% blooming. The treatment were designed as follows: Untre ted flower as control. Flowers that were sprayed by 100 units/ml of RNase that was dissolved in water or 10 mM cit ate buffer pll 3.5 (R\V and RB, res ect ively) , wi thout or wi th 0.025% Triton X-100 ( RWT and RBT , re ect ively) . Table 3 shows that RNase signi ficant ly reduced frui t set in nectarine trees. The use of a buffer in addition to Triton X-100 did not si nificantly alter the results. In plum cv. Vi son branches were sprayed wi th 1, 10 or 100 units/ml of RNase, dissolved in water (RI, R10 and R100, respectively, supplemented with 0.025% Triton X-100 and applied once at 50% blooming (I) or three t imes, starting from 10% blooming (M). Table 4 shows that the combination of high concentration of RNase and repeated applications during a n t h e s i s were most ef fect ive in reducing fruit- et. TABLE FRUTT SET OF NECTARINE CV. FANTASIA IN ROSH ZURIM Treatment Number of Total Percentage P=0.05 branches number of of fruits f 1 ower s per branch Cont ro 1 100 63.60 ±13.64 RW 81 41.80 ±8.70 RWT 89 43.75 ±7.74 RB 97 45.0 ±9.56 RBT 85 41.0 ib ±1 .16 R = Spraying wi th RN se (e tr ct from Asp rgillus niger W = Water as Solvent B = Buffer as a solvent T = Tr i ton x 100 TABLE FRUIT SET OF PLUM CV VIKSON IN HE ETZ« No. Treatment Concentration Percent of Frui t Set In of RNase Experiment Λ b Experiment B c P(.r m 1 Cont ro 1 ab 13.0 RNase 9.0 9.7 ab RNase 10 5.7 ab 7.8 ab RNase 100 3.4 a = At a certainty of P=0.05 b E periment A = Three sprayings s t ar t ing a t 10' f 1 owc i ng c Experiment B = Sin le spraying at 50% flowering. EXAMPI.F Eff c t on Shotbe i in G pe-Vines The vineyard tested did not receive any sort of spraying of gibberell in to increase the size of the grapes. The vine was sprayed by hand during flowering on 6/5/93. On 11/7/93 the bunches were weighed and the number of shot berries counted. The results are shown in Table 5. EXAMΓI.E RNase samples from the fermentation of Aspergillus niger were loaded on 15% polyacrylamide gel electrophoresis (SDS PAGE). The proteins were resolved and after the run the proteins were renatured in si tu by removing the SDS using an isopropanol buffer. After the run the gel was divided into t o lanes. One lane was subjected to RNase zymogram detect ion (overlayed on 0.1% yeast RNΛ and 1.5% agarose for several hours) and then stained wi th 0.1% toluidine blue. RNase bands were visualized as evident by a whi te halo around an act ive band at pproximately !5KDa and 30 KD . The corres onding band from the paral lel lane was di ected and inserted into peach pol len g e r i na t i ng t ube s . The perc n t o f germination and pollen tube growth were recorded after 24 hours. The results (pollen tube length) are shown in Table 6. TABLE 5 THE EFFECT OF RNASE ON SHOTBERRIES IN GRAPES" No. Treatmen t Con. No. Sho t herr i es No. Shotberries RNase Per Bunch Per lOOg Grapes contro1 77.1 45.1 RNase 1u /m1 84 46.9 RNase lOu/ l 45.5 b 33.8 ab RNase lOOu/ml 38.9 b 18.6 b a = At a ce tainty of p=0.05 TABLE POLLEN TUBE LENGTH OF RNASE SAMPLES ISOLATED FROM THE FERMENTATION OF Λ. NIGER Gel Without Bands at Un t e ted P o t e i n s 30 Dg Λver age 0. 8 a 0.664 b 0.33 c Standard 0.19 0.38 0.24 STE" 0.03 0.06 0.03 a = At p = 0.05 EXAMPLE 8 The effect on the germinat ion and the pol len tube length of peacli po l l e wi t h bo i l ed RNa se ( or i g i na t i n f rom Λ pergillu ni e wa tudied. The resul t s sho n in T bles 7 and 8 sho a m ed effect even af ter boi ling the RNase. TABLE PEACH POLLEN GERMTNATTON IN BOILED RNASE" Perc n I Ge in i i.a 1 i on Rnase Concentr tion ( uni t /ml 45.0 40 b 0.09 0.9 a = From Asper illus ni er TABLE 8 PFACII POLLEN TUBE I FNGTH IN BOILED RNASF" Pollen Tube Leng h Rnase Concentrations (mm (uni t s /ml ) 0.55 0.48 0.09 0.30 0.9 a = from Aspergillus nigcr

Claims: C L A I M S A method of the biological control of pollen in plants applying an affective amount of activated or inactivated RNase or a mixture of these. Λ method in accordance with Claim 1. wherein the inactivated RNase is chosen from the group consisting of protein, protein fragments, ribozymes, polypeptidcs and mixture of these. A method in accordance with Claim 1 or 2, wherein the RNase is obtained from sources chosen from the group consisting of bacterial, plant, animal, fungal and vir l. Λ metho in accordance with any of Claims 1 to 3, wherein the RNase i s secreted by fungal sources chosen from the group consi st ing of Λspergi l lus niger, Aspergillus cla ctur, Λ pergillus ory ae and Rhi sopu mivcu . Λ method in accordance with Claim 4, wherein the fungal sources are chosen from the group consisting of Asper illus niger Bl and Aspergillus oryzae. A m thod in accordance wi th any of Clai s 1 to 5, wherein the plant s are chosen from the group con¬ sisting of fruit trees, ornamental trees, vegetables field crops, plantations and ornamentals. 7. Λ method in accordance with any of Claims 1 to 6, wherein the t r ee s ar e chos en f rom t he group consisting of deciduous , tropical , subtropical , citrus, nut trees and grape vines. 8. A method in accordance wi th Claim 7, wherein the deciduou l an t s a r e cho s en f rom t he g roup consist ing of stone frui t , pears, a les, quince, peaches, plum, nectarines, cherries, and apricots. 9. A method in accordance wi th Claim 7, wherein the subt ropica l plant s are chosen f rom the group consisting of avocado, mango, and persimmon. 10. Λ method in accordance with Claim 7, wherein the citrus plants are chosen from the group consisting of orange, lemon, grapefruit, Clementine, mandarin, citrus, pome la and their hybrids. 11. A ethod in acco dance wi th Claim 6 , herein the field cro s are cereals. 12. A metho in accordanc wi th Claim , wherein the ornamen t a l t r ee s ar e cho s en f rom t he group consisting of olive trees and ficus sp. 13. Λ method o f pr e en t i n g t he s e l f - or c ro s s - fertilizations of plants by applying an effective amount of ac t i va t ed or Inac t i va t ed RNase or a mixture of these. 1 . A etho in accordance wi th Clai 13, herein the inactivated RNase is chosen from the group consist¬ ing of prot ein , prot ein f ragment s , ribozymes , pol ypep t i de , and mixture of these. 15. A method in accordance with Claim 13 or 14, wherein the RNase is obtained from sources chosen from the group consisting of bacterial, plant, animal, fungal and viral. 16. Λ method in accordance with any of Claims 13 to 15, wherein the RNase i s secret ed by fungal sources chosen from the grou consi s t ing of Λspergi l lus niger, A er i l lus c 1 a v e t u r , Asper i l lu ryzae, and Rhi sopus miveus . 17. A method in accordance wi th Claim 16, wherein the fungal sources are chosen from the group consisting of Λspergillus niger Bl and Λspergillus oryzae. 18. Λ method in accordance with any of Claims 13 to 17, wherein the plants are chosen from the group con¬ sisting of fruit trees, ornamental trees, vegetables field crops, plantations and ornamentals. 19. A method i ccordanc ith any of Clai s 13 to 18, wherein the t ree s are cho s en f r o t he g roup consisting of deciduous , tro ic l , subtropical , citrus, nut trees, and grape vines. 20. A method in accordance with Claim 19, wherein the deciduou s p l an t s ar e cho s en f r om t he group consist ing of stone frui t , pears, apples, quince, peaches, plum, nectarines, cherries, and apricots. 21. A method in accordance with Claim 19, wherein the subtropical plant s are cho sen from the g roup consisting of avocado, mango and persimmon. 22. A method in accordance wi th Claim 19, wherein the citrus plants are chosen from the group consisting of orange, lemon, grapefrui t , Clementine, mandarin, citrus, pome 1 a and their hybrids. 23. Λ method in accordance with Claim 18, wherein the field crops are cereals. 24. A method in accordance with Claim 18, wherein the ornamental t ees a r e c ho s en f r o t h e g r oup consistin of olive trees and fieus sp. 25. A method of thinning frui t by applying an effective amount of ac t i v ted or inac t iva t ed RNase or a ixture of these to the frui t trees during anthesis. 26. A method in accordance wi th Claim 25, wherein the inactiv a t ed RN s e i s cho s en f r om t he group consisting of protein, protein segments, ribozymes, polypeptides, and mixture of these. 27. A method in accordance with Claim 25 or 26, wherein the RNase is obtained from sources chosen from the group consi t in of bacterial, plant, animal, fungal and vi al. 28. A method in accordance with any of Claims 25 to 27, wherein the RNAase i s sec eted by fungal sources chosen from the group consi s t ing of Λspergi l lus oryzae and Rhisopus miveus. 29. Λ method in accordance with Claim 28, wherein the fungal sources are chosen from the group consisting of Aspergillus niger Bl and Aspergillus oryzae. 30. Λ method in accordance wi th any of Claims 25 to 29, wherein the plants are chosen from the group con¬ sisting of fruit trees, orna ental trees, vegetables field c-ops, plantations and ornamentals. 31. A method in accordance with ny of Claims 25 to 30, wherein the pl ant s are chosen from the group consisting of deciduous , tropical , subtropical , citrus, nut trees, and grape vines. 32. A method in accordance with Claim 31, wherein the deciduou l an t are cho s n f rom t he g roup consist ing of stone frui t , pears, apples, quince, peaches, plum, nectarines, cherries and apricots. A method in accordance with Claim 31, wherein the subtropica l plan t s are chosen from the group consisting of avocado, mango and persimmon. 34. Λ method in accordance with Claim 31, wherein the citrus plants arc chosen from the group consisting of orange, lemon, grapefruit, Clementine, mandarin, citrus, pomelo and their hybrids. 35. A method of reducing seeds in plants by applying an effective amount of activated or inactivated RNAse or a mixture of these to the plants during anthesis. 36. A method in accordance wi th Claim 35, wherein the inactiv a t ed RMΛ s e i s cho s en f rom t he g roup consisting of protein, protein segments, ribozymes, poly eptides, and mixture of these. 37. A method in accordance with Claims 35 or 36, wherein the RNAse is obtained from sources chosen from the group consisting of bacterial, plant, animal, fungal and viral. 38. Λ method in accordance with any of Claims 35 to 37, wherein the RNA i s secreted by fungal sources chosen from the group con i s t ing of As erg i l lus niger, Λ pergillus cla ctur, Λspergillus or zae and Rhi sopu miveus. 39. Λ method in accordance with Claim 38, wherein the fungal sources are chosen from this group consisting of Aspergillus niger Bl and Aspergi llus oryzae. 40. A method in accordance with any of Claims 35 to 39, wherein the pl an t are chosen f rom the group consist ing of ci trus, sub tropicals and grapes. 41. Λ method in accordance wi tli Claim 40, wherein the citrus plants are chosen from the group consisting of orange, lemon, grapefruit, Clementine, mandarin, citron, pomola and their hybrids. 42. A method in accordance with Clai 40, wherein the subt ropical plant s are chosen from the group consisting of avocado, mango and persimmon. 43 A method of reducing the incidence of shotberries in grape vines by applying an effect ive amount of activated or inactivated RNase or a mixture of these to the grape vines. 44. Λ method in accordance with Claim 43, wherein the inactiva t ed RNΛs e i s cho sen from t he grou consisting of protein, protein fragments, ribozymes, polypeptides and a mixture of these. 45. A method in accordance with Claim 43 or 44, wherein the RNAse is obtained from sources chosen from the group consisting of bacterial, plant, animal, fungal and vi Ta 1. 46. A method in accordance with any of Claims 43 to 45, whercin the RNΛsc i s secreted by fungal sources chosen from the group consi s t ing of Aspergi l lus niger, Λ pergillu clavetur, Λspergillus oryzae and Rhisopum miveus. 47. Λ method in accordance with Claim 46, wherein the fungal sources are chosen from the group consisting of Aspergillus niger Bl. and Aspergillus oryzae. 48. A method applying an effect ive amount of the RNAse to plan s via bees. 49. A method in accordance with any of Claims 1 to 48 with reference to the examples.

Inventors: BRAVDO BEN-AMI, GOREN RAPHAEL, OZERI UZI, REIZ LEVAVA, SHOSEYOV ODED, BRAVDO, BEN-AMI, GOREN, RAPHAEL, OZERI, UZI, REIZ, LEVAVA, SHOSEYOV, ODED
Applicants: BRAVDO BEN AMI, COHEN A DAVID, GOREN RAPHAEL, OZERI UZI, REIZ LEVAVA, SHOSEYOV ODED, YISSUM RES DEV CO, BRAVDO, BEN-AMI, COHEN, A., DAVID, GOREN, RAPHAEL, OZERI, UZI, REIZ, LEVAVA, SHOSEYOV, ODED, YISSUM RESEARCH DEVELOPMENT CO. OF THE HEBREW UNIV
Application year: 1994
Main classification: A61K38
IPC codes: A01H1, C12N5, C12N15